Rel Family Transcription Factor NFAT5 Upregulates COX2 via HIF-1α Activity in Ishikawa and HEC1a Cells.

Nuclear factor of activated T cells 5 (NFAT5) and cyclooxygenase 2 (COX2; PTGS2) both participate in diverse pathologies including cancer progression. However, the biological role of the NFAT5-COX2 signaling pathway in human endometrial cancer has remained elusive. The present study explored whether NFAT5 is expressed in endometrial tumors and if NFAT5 participates in cancer progression. To gain insights into the underlying mechanisms, NFAT5 protein abundance in endometrial cancer tissue was visualized by immunohistochemistry and endometrial cancer cells (Ishikawa and HEC1a) were transfected with NFAT5 or with an empty plasmid. As a result, NFAT5 expression is more abundant in high-grade than in low-grade endometrial cancer tissue. RNA sequencing analysis of NFAT5 overexpression in Ishikawa cells upregulated 37 genes and downregulated 20 genes. Genes affected included cyclooxygenase 2 and hypoxia inducible factor 1α (HIF1A). NFAT5 transfection and/or treatment with HIF-1α stabilizer exerted a strong stimulating effect on HIF-1α promoter activity as well as COX2 expression level and prostaglandin E2 receptor (PGE2) levels. Our findings suggest that activation of NFAT5-HIF-1α-COX2 axis could promote endometrial cancer progression.

The Effect of Low-Intensity Pulsed Ultrasound on Bone Regeneration and the Expression of Osterix and Cyclooxygenase-2 during Critical-Size Bone Defect Repair.

Low-intensity pulsed ultrasound (LIPUS) is a form of ultrasound that utilizes low-intensity pulsed waves. Its effect on bones that heal by intramembranous ossification has not been sufficiently investigated. In this study, we examined LIPUS and the autologous bone, to determine their effect on the healing of the critical-size bone defect (CSBD) of the rat calvaria. The bone samples underwent histological, histomorphometric and immunohistochemical analyses. Both LIPUS and autologous bone promoted osteogenesis, leading to almost complete closure of the bone defect. On day 30, the bone volume was the highest in the autologous bone group (20.35%), followed by the LIPUS group (19.12%), and the lowest value was in the control group (5.11%). The autologous bone group exhibited the highest intensities of COX-2 (167.7 ± 1.1) and Osx (177.1 ± 0.9) expression on day 30. In the LIPUS group, the highest intensity of COX-2 expression was found on day 7 (169.7 ±1.6) and day 15 (92.7 ± 2.2), while the highest Osx expression was on day 7 (131.9 ± 0.9). In conclusion, this study suggests that LIPUS could represent a viable alternative to autologous bone grafts in repairing bone defects that are ossified by intramembranous ossification.

[Ligusticum cycloprolactam inhibits IL-1ß-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway].

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

Triptolide Administration Alters Immune Responses to Mitigate Insulin Resistance in Obese States.

Individuals who are overweight or obese are at increased risk of developing prediabetes and type 2 diabetes, yet the direct molecular mechanisms that connect diabetes to obesity are not clear. Chronic, sustained inflammation is considered a strong risk factor in these interactions, directed in part by the short-lived gene expression programs encoding for cytokines and pro-inflammatory mediators. In this study, we show that triptolide administration in the C57BL/6 diet-induced obese mice at up to 10 µg/kg/day for 10 weeks attenuated the development of insulin resistance and diabetes, but not obesity, in these animals. Significant reductions in adipose tissue inflammation and improved insulin sensitivity were observed in the absence of changes in food intake, body weight, body composition, or energy expenditure. Analysis of the core cluster of biomarkers that drives pro-inflammatory responses in the metabolic tissues suggested TNF-α as a critical point that affected the co-development of inflammation and insulin resistance, but also pointed to the putatively protective roles of increased COX-2 and IL-17A signaling in the mediation of these pathophysiological states. Our results show that reduction of diet-induced inflammation confers partial protection against insulin resistance, but not obesity, and suggest the possibility of achieving overweight phenotypes that are accompanied by minimal insulin resistance if inflammation is controlled.

Methyl jasmonate ameliorates pain-induced learning and memory impairments through regulating the expression of genes involved in neuroinflammation.

OBJECTIVE: Orofacial pain with high prevalence is one of the substantial human health issues. The importance of this matter became more apparent when it was revealed that orofacial pain, directly and indirectly, affects cognition performances. Currently, researchers have focused on investigating pharmaceutics to alleviate pain and ameliorate its subsequent cognitive impairments. DESIGN: In this study, the rats were first treated with the central administration of methyl jasmonate (MeJA), which is an antioxidant and anti-inflammatory bio-compound. After 20 min, orofacial pain was induced in the rats by the injection of capsaicin in their dental pulp. Subsequently, the animals' pain behaviors were analyzed, and the effects of pain and MeJA treatments on rats learning and memory were evaluated/compared using the Morris water maze (MWM) test. In addition, the expression of tumor necrosis factor-α (TNF-α), IL-1ß, BDNF, and COX-2 genes in the rats' hippocampus was evaluated using real-time polymerase chain reaction. RESULTS: Experiencing orofacial pain resulted in a significant decline in the rats learning and memory. However, the central administration of 20 µg/rat of MeJA effectively mitigated these impairments. In the MWM, the performance of the MeJA-treated rats showed a two- to threefold improvement compared to the nontreated ones. Moreover, in the hippocampus of pain-induced rats, the expression of pro-inflammatory factors TNF-α, IL-1ß, and COX-2 significantly increased, whereas the BDNF expression decreased. In contrast, MeJA downregulated the pro-inflammatory factors and upregulated the BDNF by more than 50%. CONCLUSIONS: These findings highlight the notable antinociceptive potential of MeJA and its ability to inhibit pain-induced learning and memory dysfunction through its anti-inflammatory effect.

In Vitro Inhibition of Colorectal Cancer Gene Targets by Withania somnifera L. Methanolic Extracts: A Focus on Specific Genome Regulation.

An approach that shows promise for quickening the evolution of innovative anticancer drugs is the assessment of natural biomass sources. Our study sought to assess the effect of W. somnifera L. (WS) methanolic root and stem extracts on the expression of five targeted genes (cyclooxygenase-2, caspase-9, 5-Lipoxygenase, B-cell lymphoma-extra-large, and B-cell lymphoma 2) in colon cancer cell lines (Caco-2 cell lines). Plant extracts were prepared for bioassay by dissolving them in dimethyl sulfoxide. Caco-2 cell lines were exposed to various concentrations of plant extracts, followed by RNA extraction for analysis. By explicitly relating phytoconstituents of WS to the dose-dependent overexpression of caspase-9 genes and the inhibition of cyclooxygenase-2, 5-Lipoxygenase, B-cell lymphoma-extra-large, and B-cell lymphoma 2 genes, our novel findings characterize WS as a promising natural inhibitor of colorectal cancer (CRC) growth. Nonetheless, we recommend additional in vitro research to verify the current findings. With significant clinical benefits hypothesized, we offer WS methanolic root and stem extracts as potential organic antagonists for colorectal carcinogenesis and suggest further in vivo and clinical investigations, following successful in vitro trials. We recommend more investigation into the specific phytoconstituents in WS that contribute to the regulatory mechanisms that inhibit the growth of colon cancer cells.

Interpreting the Mechanism of Active Ingredients in Polygonati Rhizoma in Treating Depression by Combining Systemic Pharmacology and In Vitro Experiments.

Polygonati Rhizoma (PR) has certain neuroprotective effects as a homology of medicine and food. In this study, systematic pharmacology, molecular docking, and in vitro experiments were integrated to verify the antidepressant active ingredients in PR and their mechanisms. A total of seven compounds in PR were found to be associated with 45 targets of depression. Preliminarily, DFV docking with cyclooxygenase 2 (COX2) showed good affinity. In vitro, DFV inhibited lipopolysaccharide (LPS)-induced inflammation of BV-2 cells, reversed amoeba-like morphological changes, and increased mitochondrial membrane potential. DFV reversed the malondialdehyde (MDA) overexpression and superoxide dismutase (SOD) expression inhibition in LPS-induced BV-2 cells and decreased interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and IL-6 mRNA expression levels in a dose-dependent manner. DFV inhibited both mRNA and protein expression levels of COX2 induced by LPS, and the activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) and caspase1 was suppressed, thus exerting an antidepressant effect. This study proves that DFV may be an important component basis for PR to play an antidepressant role.

Xin-Yi-Qing-Fei-Tang and its critical components reduce asthma symptoms by suppressing GM-CSF and COX-2 expression in RBL-2H3 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine (TCM) XYQFT is composed of 10 herbs. According to the NHIRD, XYQFT is one of the top ten most commonly used TCM prescriptions for asthma treatment. AIM OF THE STUDY: The aim of this study was to explore whether XYQFT reduces asthma symptoms in a mouse model of chronic asthma and determine the immunomodulatory mechanism of mast cells. MATERIALS AND METHODS: BALB/c mice were intratracheally (it) stimulated with 40 µL (2.5 µg/µL) of Dermatophagoides pteronyssinus (Der p) once a week for 6 consecutive weeks and orally administered XYQFT at 1 g/kg 30 min before Der p stimulation. Airway hypersensitivity, inflammatory cells in the BALF and total IgE in the blood were assessed in mice. In addition, RBL-2H3 cells (mast cells) were stimulated with DNP-IgE, after which different concentrations of XYQFT were added for 30 min to evaluate the effect of XYQFT on the gene expression and degranulation of DNP-stimulated RBL-2H3 cells. After the compounds in XYQFT were identified using LC‒MS/MS, the PBD method was used to identify the chemical components that inhibited the expression of the GM-CSF and COX-2 genes in mast cells. RESULTS: The airway hypersensitivity assay demonstrated that XYQFT significantly alleviated Der p-induced airway hypersensitivity. Moreover, cell counting and typing of bronchoalveolar lavage fluid revealed a significant reduction in Der p-induced inflammatory cell infiltration with XYQFT treatment. ELISA examination further indicated a significant decrease in Der p-induced total IgE levels in serum following XYQFT administration. In addition, XYQFT inhibited the degranulation and expression of genes (IL-3, IL-4, ALOX-5, IL-13, GM-CSF, COX-2, TNF-α, and MCP-1) in RBL-2H3 cells after DNP stimulation. The compounds timosaponin AIII and genkwanin in XYQFT were found to be key factors in the inhibition of COX-2 and GM-CSF gene expression in mast cells. CONCLUSION: By regulating mast cells, XYQFT inhibited inflammatory cell infiltration, airway hypersensitivity and specific immunity in a mouse model of asthma. In addition, XYQFT synergistically inhibited the expression of the GM-CSF and COX-2 genes in mast cells through timosaponin AIII and genkwanin.

Unravelling the role of HAS2, GREM1, and PTGS2 gene expression in cumulus cells: implications for human oocyte development competency - a systematic review and integrated bioinformatic analysis.

The leading indicator for successful outcomes in in-vitro fertilization (IVF) is the quality of gametes in oocytes and sperm. Thus, advanced research aims to highlight the parameter in assessing these qualities - DNA fragmentation in sperm and oocyte development capacity (ODC) via evaluation of microenvironments involving its maturation process. Regarding oocytes, most evidence reveals the role of cumulus cells as non-invasive methods in assessing their development competency, mainly via gene expression evaluation. Our review aims to consolidate the evidence of GDF-9 derivatives, the HAS2, GREM1, and PTGS2 gene expression in cumulus cells used as ODC markers in relevant publications and tailored to current IVF outcomes. In addition to that, we also added the bioinformatic analysis in our review to strengthen the evidence aiming for a better understanding of the pathways and cluster of the genes of interest - HAS2, GREM1, and PTGS2 in cumulus cell level. Otherwise, the current non-invasive method can be used in exploring various causes of infertility that may affect these gene expressions at the cumulus cell level. Nevertheless, this method can also be used in assessing the ODC in various cohorts of women or as an improvement of markers following targeted tools or procedures by evaluating the advancement of these gene expressions following the targeted intervention.

Lipopolysaccharide regulation of antiinflammatory tristetraprolin family and proinflammatory gene expression in mouse macrophages.

OBJECTIVE: Tristetraprolin (TTP/ZFP36) family proteins exhibit antiinflammatory effects by destabilizing proinflammatory mRNAs. Previous studies showed that bacterial endotoxin lipopolysaccharides (LPS) stimulated TTP and tumor necrosis factor (TNF) gene expression, but less was known about LPS effects on TTP homologues and other proinflammatory gene expression in macrophages. The objective was to investigate LPS regulation of TTP family gene and TTP-targeted gene expression in mouse RAW264.7 macrophages using much higher concentrations of LPS and much longer treatment time than previous studies. RESULTS: MTT assay showed that LPS was not toxic to the cells under LPS treatment up to 1000 ng/mL for 2-24 h. LPS mildly affected the soluble protein content in the cells. qPCR assay showed that LPS stimulated TTP mRNA rapidly but not sustainably with 40, 10, and 3 fold of the DMSO control after 2, 8 and 24 h treatment, respectively. Immunoblotting confirmed qPCR results on LPS stimulation of TTP gene expression in the mouse macrophages. LPS exhibited minimal effects on ZFP36L1, ZFP36L2 and ZFP36L3 mRNA levels. LPS increased mRNA levels of TNF, COX2, GM-CSF, INFγ and IL12b up to 311, 418, 11, 9 and 4 fold, respectively. This study demonstrated that LPS did not affect macrophage viability, dramatically increased antiinflammatory TTP gene expression as well as proinflammatory TNF and COX2 gene expression but had only mild effects on TTP homologues and other proinflammatory cytokine gene expression in the mouse macrophages.

Pentraxin 3 exacerbates psoriasiform dermatitis through regulation of macrophage polarization.

OBJECTIVE: To elucidate the mechanism of Pentraxin 3 (PTX3) in the pathogenesis of psoriasiform dermatitis using Ptx3-knockout (Ptx3-KO) background mice. METHODS: An Imiquimod (IMQ)-induced murine psoriatic model was created using Ptx3-KO (Ptx3-/-) and wild-type (Ptx3+/+) mice. Skin lesion severity and expression of inflammatory mediators (IL-6 and TNFα) were assessed using PASI score and ELISA, respectively. Cutaneous tissues from the two mice groups were subjected to histological analyses, including HE staining, Masson staining, and Immunohistochemistry (IHC). The PTX3, iNOS, COX2, and Arg1 expressions were quantified and compared between the two groups. We used RNA-seq to clarify the underlying mechanisms of the disease. Flow cytometry was used to analyze systemic Th17 cell differentiation and macrophage polarization. RESULT: The psoriatic region exhibited a higher PTX3 expression than the normal cutaneous area. Moreover, PTX3 was upregulated in HaCaT cells post-TNFα stimulation. Upon IMQ stimulation, Ptx3-/- mice displayed a lower degree of the psoriasiform dermatitis phenotype compared to Ptx3+/+ mice. Consistent with the RNA-seq results, further experiments confirmed that compared to the wild-type group, the PTX3-KO group exhibited a generally lower IL-6, TNFα, iNOS, and COX2 expression and a contrasting trend in macrophage polarization. However, no significant difference in Th17 cell activation was observed between the two groups. CONCLUSIONS: This study revealed that PTX3 was upregulated in psoriatic skin tissues and TNFα-stimulated HaCaT cells. We also discovered that PTX3 deficiency in mice ameliorated the psoriasiform dermatitis phenotype upon IMQ stimulation. Mechanistically, PTX3 exacerbates psoriasiform dermatitis by regulating macrophage polarization rather than Th17 cell differentiation.

O-GlcNAc regulates anti-fibrotic genes in lung fibroblasts through EZH2.

Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-ß1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-ß1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.

CircSCUBE3 promoted ferroptosis to inhibit lung adenocarcinoma progression.

CircRNAs can regulate ferroptosis and affect cancer development and are promising biomarkers and therapeutic targets in lung cancer. circSCUBE3 is expressed in lung adenocarcinoma (LUAD) tissues. In this study, our purpose was to study the role and regulatory mechanism of circSCUBE3 in LUAD ferroptosis. circSCUBE3 was identified to be significantly downregulated in LUAD samples and cell lines. The expression of biomarkers related to lipid oxidation (4-HNE) and ferroptosis (Ptgs2) was both downregulated in LUAD tissues, suggesting the ferroptosis resistance in LUAD. Erastin, a ferroptosis inducer, was used to stimulate the LUAD cells for 48 h. The cell viability, 4-HNE and Ptgs2 level of LUAD cells were decreased by exposure to erastin while the expression of circSCUBE3 was not significantly altered. We then overexpressed circSCUBE3 in LUAD cells and found it decreased the GSH level and GSH/GSSG ratio in LUAD cells. CircSCUBE3 might serve as an independent factor of ferroptosis and may induce ferroptosis in LUAD by inhibiting GSH synthesis. The loss-of-function experiments were conducted, and circSCUBE3 deficiency reversed the erastin-induced reduction in cell viability, GSH level, GSH/GSSG ratio, mitochondrial membrane potential and elevation in MDA content, Ptgs2, 4-HNE expression as well as lipid ROS production. CircSCUBE3 negatively regulated GPX4 expression in LUAD cells, and the silencing of GPX4 counteracted the impact of circSCUBE3 deficiency on LUAD cell viability as well as ferroptosis, suggesting that circSCUBE3 regulated the GPX4-mediated GSH synthesis in LUAD. CircSCUBE3 was to bind to CREB, which activated the transcription of GPX4. CircSCUBE3 negatively regulated GPX4 expression by competitively interacting with CREB. In the tumor-bearing mouse models, circSCUBE3 silencing promoted tumor growth and reversed the erastin treatment-induced inhibition on tumorigenesis in vivo. In conclusion, circSCUBE3 inhibited LUAD development by promoting ferroptosis via the CREB/GPX4/GSH axis, which might provide a novel option for the LUAD targeted therapy.

The cyclooxygenase-2 upregulation mediates production of PGE2 autacoid to positively regulate interleukin-6 secretion in chronic rhinosinusitis with nasal polyps and polyp-derived fibroblasts.

Chronic rhinosinusitis (CRS) can be traditionally classified as CRSwNP [with nasal polyps (NPs)] and CRSsNP (without NPs) based on the clinical phenotypes but recently suggested to be classified by the endotypes. We have identified overexpression of the cyclooxygenase-2 (COX-2) gene in NP tissues of Taiwanese CRSwNP patients. Therefore, in this study, we sought to investigate its protein expression/location/distribution in NP specimens and explore its roles in nasal polyposis. The COX-2 protein and mRNA expression was found higher in NPs than that in the control and CRSsNP patients' nasal tissues, mainly located at the epithelium and subepithelial stroma. Consistently, the CRS-related peptidoglycan (PGN) and bradykinin provoked COX-2 mRNA and protein upregulation in the human NP-derived fibroblasts and caused PGE2, thromboxane A2 (TXA2), and interleukin (IL-6) secretion in culture medium. Further analysis revealed that the PI3K/Akt activation and COX-2 induction were necessarily required for PGN-induced IL-6 production/secretion and the induced PGE2, but not TXA2, was speculated to affect IL-6 protein trafficking and production. Finally, the IL-6 increase observed in vitro could also be detected in NP tissues. Collectively, we demonstrated here that COX-2 protein and IL-6 are overexpressed in human NP tissues. In response to PGN challenge, the PI3K/Akt activation and COX-2-mediated PGE2 autacoid correlates with extracellular IL-6 protein trafficking/production in NP-derived fibroblasts, which can additionally contribute to the production of Th17-related cytokines such as IL-17 and TNF-α. This study also suggests COX-2 as a special biomarker for CRSwNP endotyping and may highlight the importance of COX-2 inhibitors in treating CRSwNP.

Identification of New Markers of Angiogenic Sprouting Using Transcriptomics: New Role for RND3.

BACKGROUND: New blood vessel formation requires endothelial cells to transition from a quiescent to an invasive phenotype. Transcriptional changes are vital for this switch, but a comprehensive genome-wide approach focused exclusively on endothelial cell sprout initiation has not been reported. METHODS: Using a model of human endothelial cell sprout initiation, we developed a protocol to physically separate cells that initiate the process of new blood vessel formation (invading cells) from noninvading cells. We used this model to perform multiple transcriptomics analyses from independent donors to monitor endothelial gene expression changes. RESULTS: Single-cell population analyses, single-cell cluster analyses, and bulk RNA sequencing revealed common transcriptomic changes associated with invading cells. We also found that collagenase digestion used to isolate single cells upregulated the Fos proto-oncogene transcription factor. Exclusion of Fos proto-oncogene expressing cells revealed a gene signature consistent with activation of signal transduction, morphogenesis, and immune responses. Many of the genes were previously shown to regulate angiogenesis and included multiple tip cell markers. Upregulation of SNAI1 (snail family transcriptional repressor 1), PTGS2 (prostaglandin synthase 2), and JUNB (JunB proto-oncogene) protein expression was confirmed in invading cells, and silencing JunB and SNAI1 significantly reduced invasion responses. Separate studies investigated rounding 3, also known as RhoE, which has not yet been implicated in angiogenesis. Silencing rounding 3 reduced endothelial invasion distance as well as filopodia length, fitting with a pathfinding role for rounding 3 via regulation of filopodial extensions. Analysis of in vivo retinal angiogenesis in Rnd3 heterozygous mice confirmed a decrease in filopodial length compared with wild-type littermates. CONCLUSIONS: Validation of multiple genes, including rounding 3, revealed a functional role for this gene signature early in the angiogenic process. This study expands the list of genes associated with the acquisition of a tip cell phenotype during endothelial cell sprout initiation.

Expression of PTGS2 along with genes regulating VEGF signalling pathway and association with high-risk factors in locally advanced oral squamous cell carcinoma.

BACKGROUND: PTGS2 encodes cyclooxygenase-2 (COX-2), which catalyses the committed step in prostaglandin synthesis. Various in vivo and in vitro data suggest that COX-2 mediates the VEGF signalling pathway. In silico analysis performed in TCGA, PanCancer Atlas for head and neck cancers, demonstrated significant expression and co-expression of PTGS2 and genes that regulate VEGF signalling. This study was designed to elucidate the expression pattern of PTGS2 and genes regulating VEGF signalling in patients with locally advanced oral squamous cell carcinoma (OSCC). METHODOLOGY: Tumour and normal tissue samples were collected from patients with locally advanced OSCC. RNA was isolated from tissue samples, followed by cDNA synthesis. The cDNA was used for gene expression analysis (RT-PCR) using target-specific primers. The results obtained were compared with the in silico gene expression of the target genes in the TCGA datasets. Co-expression analysis was performed to establish an association between PTGS2 and VEGF signalling genes. RESULTS: Tumour and normal tissue samples were collected from 24 OSCC patients. Significant upregulation of PTGS2 expression was observed. Furthermore, VEGFA, KDR, CXCR1 and CXCR2 were significantly upregulated in tumour samples compared with paired normal samples, except for VEGFB, whose expression was not statistically significant. A similar expression pattern was observed in silico, except for CXCR2 which was highly expressed in the normal samples. Co-expression analysis showed a significant positive correlation between PTGS2 and VEGF signalling genes, except for VEGFB which showed a negative correlation. CONCLUSION: PTGS2 and VEGF signalling genes are upregulated in OSCC, which has a profound impact on clinical outcomes.

Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis).

BACKGROUND: Early embryonic mortality is one of the major intriguing factors of reproductive failure that causes considerable challenge to the mammalian cell biologists. Heat stress is the major factor responsible for reduced fertility in farm animals. The present study aimed to investigate the influence of heat stress on prostaglandin production and the expression of key genes, including COX-2, PGES, PGFS, ITGAV and LGALS15, in buffalo endometrial epithelial cells. METHODS AND RESULTS: Buffalo genitalia containing ovaries with corpus luteum (CL) were collected immediately post-slaughter. The stages of the estrous cycle were determined based on macroscopic observations of the ovaries. Uterine lumens of the mid-luteal phase (days 6-10 of the estrous cycle) were washed and treated with trypsin to isolate epithelial cells, which were then cultured at control temperature (38.5 °C for 24 h) or exposed to elevated temperatures [38.5 °C for 6 h, 40.5 °C for 18 h; Heat Stressed (HS)]. The supernatant and endometrial epithelial cells were collected at various time points (0, 3, 6, 12, and 24 h) from both the control and treatment groups. Although heat stress (40.5 °C) significantly (P < 0.05) increased COX-2, PGES, and PGFS transcripts in epithelial cells but it did not affect the in vitro production of PGF2α and PGE2. The expression of ITGAV and LGALS15 mRNAs in endometrial epithelial cells remained unaltered under elevated temperature conditions. CONCLUSION: It can be concluded that elevated temperature did not directly modulate prostaglandin production but, it promoted the expression of COX-2, PGES and PGFS mRNA in buffalo endometrial epithelial cells.

Neuroprotective potential of pyrazole benzenesulfonamide derivative T1 in targeted intervention against PTZ-induced epilepsy-like condition in in vivo zebrafish model.

Epilepsy is a chronic neurological disease characterized by a persistent susceptibility to seizures. Pharmaco-resistant epilepsies, impacting around 30 % of patients, highlight the urgent need for improved treatments. Neuroinflammation, prevalent in epileptogenic brain regions, is a key player in epilepsy, prompting the search for new mechanistic therapies. Hence, in this study, we explored the anti-inflammatory potential of pyrazole benzenesulfonamide derivative (T1) against pentylenetetrazole (PTZ) induced epilepsy-like conditions in in-vivo zebrafish model. The results from the survival assay showed 79.97 ± 6.65 % at 150 µM of T1 compared to PTZ-group. The results from reactive oxygen species (ROS), apoptosis and histology analysis showed that T1 significantly reduces cellular damage due to oxidative stress in PTZ-exposed zebrafish. The gene expression analysis and neutral red assay results demonstrated a notable reduction in the inflammatory response in zebrafish pre-treated with T1. Subsequently, the open field test unveiled the anti-convulsant activity of T1, particularly at a concentration of 150 µM. Moreover, both RT-PCR and immunohistochemistry findings indicated a concentration-dependent potential of T1, which inhibited COX-2 in zebrafish exposed to PTZ. In summary, T1 protected zebrafish against PTZ-induced neuronal damage, and behavioural changes by mitigating the inflammatory response through the inhibition of COX-2.

Integrating network pharmacology and renal metabonomics to reveal the protective mechanism of resveratrol on gouty nephropathy.

Resveratrol (Res) has been demonstrated to have beneficial effects on gouty nephropathy (GN). However, the mechanisms of Res on GN remain unclear. This study aimed to investigate the mechanisms of Res on GN. In this study, network pharmacology technology was used to predict the Res targets in the prevention and treatment of GN. Renal metabonomics was used to identify differential metabolites in kidney tissue of GN model rats. Finally, molecular docking technology was used to verify the binding ability of Res to key targets. Metabonomics analysis showed that 24 potentially important metabolites were involved in the prevention and treatment of GN with Res. After exposure to Res, metabolite levels normalized. The network pharmacology analysis showed that 24 key targets were involved in the prevention and treatment of GN disease. According to the metabolite-gene network diagram, we identified two core genes, PTGS1 and PTGS2, and found that both were involved in the arachidonic acid metabolism pathway. Molecular docking further verified the affinity of Res binding to PTGS1 and PTGS2. In conclusion, the mechanism of Res against GN may be the regulation of arachidonic acid metabolism through the regulation of PTGS 1 and PTGS 2.

Antioxidative and Antibacterial Hydro-Ethanolic Fraction from an Asian Edible Mushroom Lentinus sajor-caju (Agaricomycetes) Suppresses Inflammatory Responses by Downregulating COX-2 and iNOS Expression.

Mushrooms are prevalently important sources of pharmaceutically active metabolites. Various mushroom species belonging to the Lentinus genus are recognized for their nutritional and therapeutic properties. One such species is L. sajor-caju, which is renowned in Southeast Asian nations for its culinary value. The primary goal of this study is to investigate the potential medicinal properties of L. sajor-caju, specifically its antioxidant, antibacterial, and anti-inflammatory effects. A hydroethanolic extract was formulated using dried basidiocarps, which exhibited a high phenolic content of approximately 14% and a flavonoid content of approximately 2.7%. The extract demonstrated significant antioxidant potential in in vitro reactions. The extract is sufficiently capable of scavenging free radicals (DPPH and ABTS) and chelate Fe2+ with EC50 values spanning from 186 to 390 µg/mL. In addition, considerable antimicrobial activity against tested pathogenic microorganisms was observed, as indicated by low MIC50 values (256-358 µg/mL). Moreover, the fraction was found to prevent heat-induced protein denaturation which signifies its anti-inflammatory potential. When tested on the RAW 264.7 cell line, reduction in the nitrite production, and downregulation of COX-2 and iNOS mRNA expression was observed which are the key regulator of inflammatory signalling systems. The study, therefore, recommends the use of L. sajor-caju in the medical and pharmaceutical industries for the benefit of humanity.